Synthetic oligonucleotides as inducers of erythroid differentiation

ABSTRACT

The invention refers to a synthetic double-stranded oligonucleotide having a length comprised between 10 and 50 bases and a nucleic acid sequence selected from the group consisting of: (a) sequences corresponding to a selected portion of the promoter of human γ-globin gene; and (b) sequences corresponding to a selected portion of the human genomic region comprised between the γ-globin gene and the δβ-cluster, for use as an inducer of erythroid differentiation.

[0001] The present invention relates to the use of syntheticoligonucleotides which are capable of inducing erythroid differentiationfor the manufacture of a medicament for the therapeutic treatment ofβ-thalassemia and neoplastic diseases, and to a pharmaceuticalcomposition including at least one of the said oligonucleotides and apharmaceutically acceptable carrier.

[0002] The existence of compounds which are able to induce the synthesisof fetal haemoglobin (HbF) in adults is known since long (1-7).

[0003] These compounds, herein referred to as “biological responsemodifiers”, are able to activate the transcription of embryonic andfetal globin genes and to induce erythroid differentiation.

[0004] In human adults, the activation of the transcription of γ-globingenes leads to the production of HbF, which mimicks a HPFH (HighPersistance of Fetal Hemoglobin) phenotype; this could reduce theseverity of β-thalassemia in affected patients (8). Accordingly, recentstudies have been focused on the search of compounds able to stimulateγ-globin gene expression at high levels, in an attempt to reducetransfusions in β-thalassemia patients (12, 13).

[0005] In addition, as it has been recently described (9, 10), acombined treatment with different biological response modifiers couldlead to a further increase of the expression of γ-globin genes.

[0006] It is further known (11) that the treatment of neoplastic cellswith compounds which are able to induce differentiation could be ofinterest in the therapy of some neoplastic diseases.

[0007] The object of the present invention is to find new biologicalresponse modifiers to be proposed for the treatment of β-thalassemiaand/or neoplastic diseases, which exhibit low toxicity in vivo and ahigh level of induction of γ-globin gene expression.

[0008] Molecules which are able to induce differentiation, exhibitingonly minor cytotoxic effects in vivo, could in fact reduce side effectswhen administered to patients in clinical trials.

[0009] The present inventors have unexpectedly found thatdouble-stranded oligonucleotides having a nucleic acid sequencecorresponding to portions of the β-like gene cluster, in particular tosome sequences of the promoter of the human γ-globin gene and to somesequences comprised between the γ-globin gene and the δβ-gene region,show the said activity.

[0010] The present invention therefore provides a syntheticdouble-stranded oligonucleotide having a nucleic acid sequence selectedfrom the group consisting of:

[0011] a) sequences corresponding to a selected portion of the promoterof human γ-globin gene; and

[0012] b) sequences corresponding to a selected portion of the humangenomic region comprised between the γ-globin gene and the δβ-cluster,

[0013] for use as an inducer of erythroid differentiation.

[0014] The nucleic acid sequence of the promoter of human γ-globin isSEQ. ID NO:1 and the nucleic acid sequence of the human genomic regioncomprised between the γ-globin gene and the δβ-cluster is SEQ. ID NO:2.

[0015] Advantageously, the double-stranded oligonucleotide of theinvention has a length comprised between 10 and 50 bases, preferablybetween 10 and 30 bases, and a nucleic acid sequence corresponding to aselected portion of SEQ. ID NO:1 or SEQ. ID NO:2.

[0016] Preferably, the double-stranded oligonucleotide of the inventionhas a nucleic acid sequence selected within the portion of SEQ. ID NO:1comprised between positions 220 and 290.

[0017] In the present description, the term “oligonucleotide” is meantto include also an oligonucleotide wherein the backbone has beenmodified according to the approaches commonly used for improvingoligonucleotides' properties such as cellular uptake, target bindingand/or stability. Such modifications include for example themodification of the linkage between the base and/or the sugar moieties.Among the current derivatives and/or techniques available for improvingthe oligonucleotides performances are the conjugation with lipophilicmoieties, chimeric technology, peptide nucleic acids, aptamers.

[0018] As it will be further illustrated in the example, it has alsobeen found that double-stranded oligonucleotides having a nucleic acidsequence as set forth in SEQ. ID NO:3, SEQ. ID NO:4 (corresponding toselected regions of the promoter of the human γ-globin gene), SEQ. IDNO:5 and SEQ. ID NO:6 (corresponding to selected regions comprisedbetween the human γδβ-globin gene cluster are particularly suitable forinducing a high expression of γ-globin genes.

[0019] Therefore, the nucleic acid sequence of the double-strandedoligonucleotide of the invention is more preferably selected from thegroup consisting of SEQ. ID No:3, SEQ. ID NO: 4, SEQ. ID No:5 and SEQ.ID NO: 6.

[0020] The synthetic oligonucleotides of the invention are able to actby mimicking human γ-globin gene regulatory sequences and by potentiallyinteracting with nuclear proteins, including transcription factors.

[0021] The person skilled in the art knows how to obtain a doublestranded oligonucleotide; for example it may be obtained by synthesisinga single-stranded oligonucleotide and then specifically annealing thesingle-stranded oligonucleotide with its complementary strand by formingWatson-Crick hydrogen bonds.

[0022] The annealing between the two complementary strands may beobtained for example by incubating the DNA in a solution of 150 mM NaClat room temperature for about two hours. Methods for the synthesis ofsingle-stranded oligonucleotides are found for example in (20).

[0023] In one embodiment of the invention, the synthetic double strandedoligonucleotide is used as an inducer of erythroid differentiation incombination with a second biological response modifier, preferablyselected from the group consisting of cytosine arabinoside, retinoicacid, plicamycin, chromomycin, hydroxyurea, guanosine triphosphate(GTP), guanosine diphosphate (GDP), and guanosine monophosphate (GMP).Cytosine arabinoside and retinoic acid are more preferred.

[0024] Also within the scope of the invention is the use of a syntheticdouble stranded oligonucleotide as previously defined, eventually incombination with a second biological response modifier as previouslydefined, for the manufacture of a medicament for the treatment ofβ-thalassemia and/or neoplastic diseases.

[0025] The present invention also provides a pharmaceutical compositioncomprising at least a synthetic double stranded oligonucleotide aspreviously defined, eventually in combination with a second biologicalresponse modifier as previously defined, and a pharmaceuticallyacceptable carrier.

[0026] The activity of the double stranded synthetic oligonucleotides ofthe invention as inducers of erythroid differentiation has been assessedby determining the level of erythroid differentiation induced in a humancultured cell line, as reported in Table 1.

[0027] The following example is provided by way of illustration only andis not intended to limit in any way the scope of the invention.

EXAMPLE

[0028] The biological activity of the double stranded oligonucleotidesrepresented by SEQ. ID NO: 3, SEQ. ID NO: 4, SEQ. ID NO: 5 and SEQ. IDNO: 6 has been evaluated by determining the ability of theseoligonucleotides to induce erythroid differentiation of the humanerythroleukemia K562 cell line, which is able to undergo erythroiddifferentiation and to increase γ-globin gene expression followingtreatment with a suitable biological response modifier (14-17). Thelevel of erythroid differentiation has been evaluated by thebenzidine-staining (16). The production of haemoglobin has beenevaluated by cellulose acetate gel electrophoresis of cytoplasmicextracts and benzidine staining of the gels (16). The expression ofγ-globin genes has been evaluated by RT-PCR (reverse transcriptase PCR)(18).

[0029] These assays have been performed after 6 days of induction withthe above indicated double stranded oligonucleotides. TABLE 1 Optimal^(a)Erythroid Oligonucleotide Concentration differentiation (SEQ. IDNOs) (μg/ml) (%) 3 10 55 4 10 40 5 10 57 6 10 38

[0030] In order to analyse hemoglobin production by erythroid inducedK562 cells, 2 μl of total fresh post-mitochondrial cell lysates wereelectrophoresed on cellulose acetate strips inTris-ethylenediamine-tetraacetic acid (EDTA)-borate buffer. After anelectrophoresis of 30 min at 5 mA, the gels were stained withbenzidine/hydrogen peroxide (1% benzidine in 4.3 M acetic acid, 3% H₂O₂)and photographed. The data obtained show that the Hb produced followingerythroid induction is mainly Hb Portland. Quantitative real-time PCRassay (21) of γ-globin mRNA transcripts was carried out with the use ofgene-specific double fluorescently labeled probes in a 7700 SequenceDetector (PE Biosystems, Warrington Cheshire, UK). The following primerand probe sequences were used for real-time PCR: γ-globin forwardprimer, 5′-TGG CAA GAA GGT GCT GAC TTC-3′ (SEQ. ID NO:7); γ-globinreverse primer, 5′-TCA CTC AGC TGG GCA AAG G-3′ (SEQ. ID. NO:8);γ-globin probe, 5′-FAM-TGG GAG ATG CCA TAA AGC ACC TGG-TAMRA-3′ (SEQ. IDNO:9), where the fluorescent reporter FAM and the quencher TAMRA are6-carboxy fluorescein (FAM) and 6-carboxy-N,N,N′,N′-tetramethylrhodamine(TAMRA) respectively. The results obtained give evidence for an increaseof γ-globin mRNA production. For instance, the oligonucleotidecorresponding to SEQ. ID NO:3 induced a nine-fold increase of γ-globinmRNA production with respect to uninduced K562 cells.

[0031] The high level of biological activity and the expected low levelof in vivo cytotoxicity of the oligonucleotides of the invention allowto propose these molecules as promising candidates for use in thedevelopment of pharmacological approaches for the treatment ofβ-thalassemia and/or neoplastic diseases.

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1 9 1 331 DNA Homo sapiens 1 aagtcctggt atcttctatg gtgggagaag aaaactagctaaagggaaga ataaattaga 60 gaaaaattgg aatgactgaa tcggaacaag gcaaaggctataaaaaaaat taagcagcag 120 tatcctcttg ggggcccctt ccccacacta tctcaatgcaaatatctgtc tgaaacggtt 180 cctggctaaa ctccacccat gggttggcca gccttgccttgaccaatagc cttgacaagg 240 caaacttgac caatagtctt agagtatcca gtgaggccaggggccggcgg ctggctaggg 300 atgaagaata aaaggaagca cccttcagca g 331 2 13700DNA Homo sapiens 2 ctcttgccca tgattcagag ctttcaagga taggctttattctgcaagca atacaaataa 60 taaatctatt ctgctgagag atcacacatg attttcttcagctctttttt ttacatcttt 120 ttaaatatat gagccacaaa gggtttatat tgagggaagtgtgtatgtgt atttctgcat 180 gcctgtttgt gtttgtggtg tgtgcatgct cctcatttatttttatatga gatgtgcatt 240 ttgatgagca aataaaagca gtaaagacac ttgtacacgggagttctgca agtgggagta 300 aatggtgttg gagaaatccg gtgggaagaa agacctctataggacaggac ttctcagaaa 360 cagatgtttt ggaagagatg ggaaaaggtt cagtgaagacctgggggctg gattgattgc 420 agctgagtag caaggatggt tcttaatgaa gggaaagtgttccaagcttt aggaattcaa 480 ggtttagtca ggtgtagcaa ttctatttta ttaggaggaatactatttct aatggcactt 540 agcttttcac agcccttgtg gatgcctaag aaagtgaaattaatcccatg ccctcaagtg 600 tgcagattgg tcacagcatt tcaagggaga gacctcattgtaagactctg ggggaggtgg 660 ggacttaggt gtaagaaatg aatcagcaga ggctcacaagtcagcatgag catgttatgt 720 ctgagaaaca gaccagcact gtgagatcaa aatgtagtgggaagaatttg tacaacatta 780 attggaaggt ttacttaatg gaatttttgt atagttggatgttagtgcat ctctataagt 840 aagagtttaa tatgatggtg ttacggacct ggtgtttgtgtctcctcaaa attcacatgc 900 tgaatcccca actcccaact gaccttatct gtgggggaggcttttgaaaa gtaattaggt 960 ttagctgagc tcataagagc agatccccat cataaaattattttccttat cagaagcaga 1020 gagacaagcc atttctcttt cctcccggtg aggacacagtgagaagtccg ccatctgcaa 1080 tccaggaaga gaaccctgac cacgagtcag ccttcagaaatgtgagaaaa aactctgttg 1140 ttgaagccac ccagtctttt gtattttgtt atagcaccttacactgagta aggcagatga 1200 agaaggagaa aaaaataagc ttgggttttg agtgaactacagaccatgtt atctcaggtt 1260 tgcaaagctc ccctcgtccc ctatgtttca gcataaaatacctactctac tactctcatc 1320 tataagaccc aaataataag cctgcgccct tctctctaactttgatttct cctattttta 1380 cttcaacatg ctttactcta gccttgtaat gtctttacatacagtgaaat gtaaagttct 1440 ttattctttt tttctttctt tcttttttct cctcagcctcagaatttggc acatgccctt 1500 ccttctttca ggaacttctc caacatctct gcctggctccatcatatcat aaaggtccca 1560 cttcaaatgc agtcactacc gtttcaggat atgcactttctttctttttt gttttttgtt 1620 ttttttaagt caaagcaaat ttcttgagag agtaaagaaataaacgaatg actactgcat 1680 aggcagagca gccccgaggg ccgctggttg ttccttttatggttatttct tgatgatatg 1740 ttaaacaagt tttggattat ttatgccttc tctttttaggccatataggg taactttctg 1800 acattgccat ggcatgtttc ttttaattta atttactgttaccttaaatt caggggtaca 1860 cgtacaggat atgcaggttt gttttatagg taaaagtgtgccatggtttt aatgggtttt 1920 ttttttcttg taaagttgtt taagtttctt gtttactctggatattggcc tttgtcagaa 1980 gaatagattg gaaaatcttt ttcccattct gtagattgtctttcgctctg atggtagttt 2040 cttttgctga gcaggagctc tttagtttaa ttagattccattggtcaatt tttgcttttg 2100 ctgcaattgc ttttcacgct ttcatcatga aatctgtgcccgtgtttata tcatgaatag 2160 tattgccttg atttttttct aggcttttta tagtttggggtttttcattt aagtctctaa 2220 tccatccgga gttaattttg gataaggtat aaggaaggagtccagtttca tttttcagca 2280 tatggctagc cagttctccc ccatcattta ttaaattgaaaatcctttcc ccattgcttg 2340 cttttgtcag gtttctaaaa gacagatggt tgtaggtacaatatgcagtt tcttcaagtc 2400 atataatacc atctgaaatc tcttattaat tcatttcttttagtatgtat gctggtctcc 2460 tctgctcact atagtgaggg caccattagc cagagaatctgtctgtctag ttcatgtaag 2520 attctcagaa ttaagaaaaa tggatggcat atgaatgaaacttcatggat gacatatgga 2580 atctaatgtg tatttgttga attaatgcat aagatgcaacaagggaaagg ttgacaactg 2640 cagtgataac ctggtattga tgatataaga gtctatagatcacagtagaa gcaataatca 2700 tggaaaacaa ttggaaatgg ggaacagcca caaacaagaaagaatcaata ctaccaggaa 2760 agtgactgca ggtcactttt cctggagcgg gtgagagaaaagtggaagtt gcagtaactg 2820 ccgaattcct ggttggctga tggaaagatg gggcaactgttcactggtac gcagggtttt 2880 agatgtatgt acctaaggat atgaggtatg gcaatgaacagaaattcttt tgggaatgag 2940 ttttagggcc attaaaggac atgacctgaa gtttcctctgaggccagtcc ccacaactca 3000 atataaatgt gtttcctgca tatagtcaaa gttgccacttctttttcttc atatcatcga 3060 tctctgctct taaagataat cttggttttg cctcaaactgtttgtcacta caaactttcc 3120 ccatgttcct aagtaaaaca ggtaactgcc tctcaactatatcaagtaga ctaaaatatt 3180 gtgtctctaa tatcagaaat tcagctttaa tatattgggtttaactcttt gaaatttaga 3240 gtctccttga aatacacatg ggggtgattt cctaaactttatttcttgta aggatttatc 3300 tcaggggtaa cacacaaacc agcatcctga acctctaagtatgaggacag taagccttaa 3360 gaatataaaa taaactgttc ttctctctgc cggtggaagtgtgccctgtc tattcctgaa 3420 attgcttgtt tgagacgcat gagacgtgca gcacatgagacacgtgcagc agcctgtgga 3480 atattgtcag tgaagaatgt ctttgcctga ttagatataaagacaagtta aacacagcat 3540 tagactatag atcaagcctg tgccagacac aaatgacctaatgcccagca cgggccacgg 3600 aatctcctat cctcttgctt gaacagagca gcacacttctcccccaacac tattagatgt 3660 tctggcataa ttttgtagat atgtaggatt tgacatggactattgttcaa tgattcagag 3720 gaaatctcct ttgttcagat aagtacactg actactaaatggattaaaaa acacagtaat 3780 aaaacccagt tttcccctta cttccctagt ttgtttcttattctgctttc ttccaagttg 3840 atgctggata gaggtgttta tttctattct aaaaagtgatgaaattggcc gggcgcggtg 3900 gctcacacct gtaatcccag cactttggga ggctgaggtgggcggatcac gaggtcagga 3960 gatcaagacc atcctggcta acatggtgaa accccatctctactaaaaat acaaaaaatt 4020 agccagagac ggtggcgggt gcctgtagtc ccagctactcgggaggctga ggcaggagaa 4080 tggcgtgaac ctgggaggca gagctgcagt gagcagagatcgcgccactg cacactccag 4140 cctgggtgac aaagcgagac tccatctcaa aaaaaaaaaaaaaaaaaaaa agaaagaaag 4200 aaagaaaaaa aaagtgatga aattgtgtat tcaatgtagtctcaagagaa ttgaaaacca 4260 agaaaggctg tggcttcttc cacataaagc ctggatgaataacaggataa cacgttgtta 4320 cattgtcaca actcctgatc caggaattga tggctaagatattcgtaatt cttatccttt 4380 tcagttgtaa cttattccta tttgtcagca ttcaggttattagcggctgc tggcgaagtc 4440 cttgagaaat aaactgcaca ctggatggtg ggggtagtgtaggaaaatgg aggggaagga 4500 agtaaagttt caaattaagc ctgaacagca aagttcccctgagaaggcca cctggattct 4560 atcagaaact cgaatgtcca tcttgcaaaa cttccttgcccaaaccccac ccctggagtc 4620 acaacccacc cttgaccaat agattcattt cactgagggaggcaaagggc tggtcaatag 4680 attcatttca ctgggagagg caaagggctg ggggccagagaggagaagta aaaagccaca 4740 catgaagcag caatgcaggc atgcttctgg ctcatctgtgatcaccagga aactcccaga 4800 tctgacactg tagtgcattt cactgctgac aagaaggctgctgccaccag cctgtgaagc 4860 aaggttaagg tgagaaggct ggaggtgaga ttctgggcaggtaggtactg gaagccggga 4920 caaggtgcag aaaggcagaa agtgtttctg aaagagggattagcccgttg tcttacatag 4980 tctgactttg cacctgctct gtgattatga ctatcccacagtctcctggt tgtctaccca 5040 tggacctaga ggtactttga aagttttgga tatctgggctctgactgtgc aataatgggc 5100 aaccccaaag tcaaggcaca tggcaagaag gtgctgatctccttcggaaa agctgttatg 5160 ctcacggatg acctcaaagg cacctttgct acactgagtgacctgcactg taacaagctg 5220 cacgtggacc ctgagaactt cctggtgagt agtaagtacactcacgcttt cttctttacc 5280 cttagatatt tgcactatgg gtacttttga aagcagaggtggctttctct tgtgttatga 5340 gtcagctatg ggatatgata tttcagcagt gggattttgagagttatgtt gctgtaaata 5400 acataactaa aatttggtag agcaaggact atgaataatggaaggccact taccatttga 5460 tagctctgaa aaacacatct tataaaaaat tctggccaaaatcaaactga gtgttttgga 5520 tgagggaaca gaagttgaga tagagaaaat aacatctttcctttggtcag cgaaattttc 5580 tataaaaatt aatagtcact tttctgcata gtcctggaggttagaaaaag atcaactgaa 5640 caaagtagtg ggaagctgtt aaaagaggat tgtttccctccgaatgatga tggtatactt 5700 ttgtacgcat ggtacaggat tctttgttat gagtgtttgggaaaattgta tgtatgtatg 5760 tatgtatgtg atgactgggg acttatccta tccattactgttccttgaag tactattatc 5820 ctacttttta aaaggacgaa gtctctaaaa aaaaaatgaaacaatcacaa tatgttgggg 5880 tagtgagttg gcatagcaag taagagaagg ataggacacaatgggaggtg cagggctgcc 5940 agtcatattg aagctgatat ctagcccata atggtgagagttgctcaaac tctggtcaaa 6000 aaggatgtaa gtgttatatc tatttactgc aagtccagcttgaggccttc tattcactat 6060 gtaccatttt cttttttatc ttcactccct ccccagctcttaggcaacgt gatattgatt 6120 gttttggcaa cccacttcag cgaggatttt accctacagatacaggcttc ttggcagtaa 6180 ctaacaaatg ctgtggttaa tgctgtagcc cacaagaccactgagttccc tgtccactat 6240 gtttgtacct atgtcccaaa atctcatctc ctttagatgggggaggttgg ggagaagagc 6300 agtatcctgc ctgctgattc agttcctgca tgataaaaatagaataaaga aatatgctct 6360 ctaagaaata tcattgtact ctttttctgt ctttatattttaccctgatt cagccaaaag 6420 gacgcactat ttctgatgga aatgagaatg ttggagaatgggagtttaag gacagagaag 6480 atactttctt gcaatcctgc aagaaaagag agaactcgtgggtggattta gtggggtagt 6540 tactcctagg aaggggaaat cgtctctaga ataagacaatgtttttacag aaagggaggt 6600 caatggaggt actctttgga ggtgtaagag gattgttggtagtgtgtaga ggtatgttag 6660 gactcaaatt agaagttctg tataggctat tatttgtatgaaactcagga tatagctcat 6720 ttggtgactg cagttcactt ctacttattt taaacaacatattttttatg atttataatg 6780 aagtggggat ggggcttcct agagaccaat caagggccaaaccttgaact ttctcttaac 6840 gtcttcaatg gtattaatag agaattatct ctaaggcatgtgaactggct gtcttggttt 6900 tcatctgtac ttcatctgct acctctgtga cctgaaacatatttataatt ccattaagct 6960 gtgcatatga tagatttatc atatgtattt tccttaaaggatttttgtaa gaactaattg 7020 aattgatacc tgtaaagtct ttatcacact acccaataaataataaatct ctttgttcag 7080 ctctctgttt ctataaatat gtacaagttt tattgtttttagtggtagtg attttattct 7140 ctttctatat atatacacac acatgtgtgc attcataaatatatacaatt tttatgaata 7200 aaaaattatt agcaatcaat attgaaaacc actgatttttgtttatgtga gcaaacagca 7260 gattaaaagg ctgagattta ggaaacagca cgttaagtcaagttgataga ggagaatatg 7320 gacatttaaa agaggcagga tgatataaaa ttagggaaactggatgcaga gaccagatga 7380 agtaagaaaa atagctatcg ttttgagcaa aaatcactgaagtttcttgc atatgagagt 7440 gacataataa atagggaaac gtagaaaatt gattcacatgtatatatata tatagaactg 7500 attagacaaa gtctaacttg ggtatagtca gaggagcttgctgtaattat attgaggtga 7560 tggataaaga actgaagttg atggaaacaa tgaagttaagaaaaaaaatc gagtaagaga 7620 ccattgtggc agtgattgca cagaactgga aaacattgtgaaacagagag tcagagatga 7680 cagctaaaat ccctgtctgt gaatgaaaag aaggaaatttattgacagaa cagcaaatgc 7740 ctacaagccc cctgtttgga tctggcaatg aacgtagccattctgtggca atcacttcaa 7800 actcctgtac ccaagaccct taggaagtat gtagcaccctcaaacctaaa acctcaaaga 7860 aagaggtttt agaagatata ataccctttc ttctccagtttcattaatcc caaaacctct 7920 ttctcaaagt atttcctcta tgtgtccacc ccaaagagctcacctcacca tatctcttga 7980 gtgggagcac atagataggc ggtgctacca tctaacagcttctgaaattc ctttgtcata 8040 tttttgagtc cccactaata acccacaaag cagaataaataccagttgct catgtacaat 8100 aatcactcaa ctgctgtctt gtagcataca ttaattaagcacattctttg aataattact 8160 gtgtccaaac aatcacactt taaaatctca cacttgtgctatcccttgcc cttctgaatg 8220 tcactctgta ttttaaatga agagatgagg gttgaatttcctgtgttact tattgttcat 8280 ttctcgatga ggagttttca cattcacctt tactggaaaacacataagta cacatcttac 8340 aggaaaaata taccaaactg acatgtagca tgaatgcttgtgcatgtagt catataaaat 8400 cttgtagcaa tgtaaacatt ctctgatata cacatacagatgtgtctata tgtctacaca 8460 atttcttatg ctccatgaac aaacattcca tgcacacataagaacacaca ctgttacaga 8520 tgcatacttg agtgcattga caaaattacc ccagtcaatctagagaattt ggatttctgc 8580 atttgactct gttagctttg tacatgctgt tcatttactctgggtgatgt ctttccctca 8640 ttttgccttg tctatcttgt actcatactt taagtcctaacttatatgtt atctcaacta 8700 agaagctatt tttttttaat tttaactggg cttaaagccctgtctataaa ctctgctaca 8760 attatgggct ctttcttata atatttagtg tttttcctactaatgtactt aatctgctca 8820 ttgtatattc ctaccactaa attttaacct cttttatggtagagacattg tcttgtaaac 8880 tcttatttcc ctagtatttg gagatgaaaa aaaagattaaattatccaaa attagatctc 8940 tcttttctac attatgagta ttacactatc catagggaagtttgtttgag acctaaactg 9000 aggaaccttt ggttctaaaa tgactatgtg atatcttagtatttataggt catgaggttc 9060 cttcctctgc ctctgctata gtttgattag tcagcaagcatgtgtcatgc atttattcac 9120 atcagaattt catacactaa taagacatag tatcagaagtcagtttatta gttatatcag 9180 ttagggtcca tcaaggaaag gacaaaccat tatcagttactcaacctaga attaaataca 9240 gctcttaata gttaattatc cttgtattgg aagagctaaaatatcaaata aaggacagtg 9300 cagaaatcta gatgttagta acatcagaaa acctcttccgccattaggcc tagaagggca 9360 gaaggagaaa atgtttatac caccagagtc cagaaccagagcccataacc agaggtccac 9420 tggattcagt gagctagtgg gtgctccttg gagagagccagaactgtcta atgggggcat 9480 caaagtatca gccataaaaa accataaaaa agactgtctgctgtaggaga tccgttcaga 9540 gagagagaga gaccagaaat aatcttgctt atgctttccctcagccagtg tttaccattg 9600 cagaatgtac atgcgactga aagggtgagg aaacctgggaaatgtcagtt cctcaaatac 9660 agagaacact gagggaagga tgagaaataa atgtgaaagcagacatgaat ggtaattgac 9720 agaaggaaac taggatgtgt ccagtaaatg aataattacagtgtgcagtg attattgcaa 9780 tgattaatgt attgataaga taatatgaaa acacagaattcaaacagcag tgaactgaga 9840 ttagaattgt ggagagcact ggcatttaag aatgtcacacttagaatgtg tctctaggca 9900 ttgttctgtg catatatcat ctcaatattc attatctgaaaattatgaat taggtacaaa 9960 gctcaaataa tttatttttt caggttagca agaacttttttttttttttt ttctgagatg 10020 gagcattgct atggttgccc aggctggagt gcaatggcatgatccaggct cactgcaaca 10080 tctgcctccc aggttcaagc gattctcctg cctcagcctcccaagtagct ggcattacag 10140 gcatgtgcca ccaccatgcc tggctaattt tctatttttagtagataggg ggtttcacca 10200 tgttggtcag gctgatctcg aactcctaac atcaggtgatccaccctcct cggcctctga 10260 atgtactggg atcacaggcg tgagccacca cacccagccaagaatgtgaa ttttgtagaa 10320 ggatataacc catatttctc tgaccctaga gtccttagtatacctcccat accatgtggc 10380 tcatcctcct tacatacatt tcccatcttt caccctaccttttccttttt gtttcagctt 10440 ttcactgtgt gtcaaaatct agaaccttat ctcctacctgctctgaaacc aacagcaagt 10500 tgacttccat tctaacccac attggcatta cactaattaaaatcgatact gagttctaaa 10560 atcatctggg attttgggga ctatgtctta cttcatacttccttgagatt tcacattaaa 10620 tgttggtgtt cattaaaggt ccttcattta actttgtattcatcacactc ttggattcac 10680 agttatatct aaactcttat atatagcctg tataatcccaattcccaagt ctgatttcta 10740 acctctgacc tccaacctca gtgccaaacc catatatcaaacaatgtact gggcttattt 10800 atatagatgt cctataggca cctcagactc agcatgggtatttcacttgt tatactaaaa 10860 ctgtttctct tccagtgttt tccattttag tcattagatagctacttgcc cattcaccaa 10920 ggtcacagat taaaatcatt tccctacctc taatcaacagttcaattctg cttcaatttg 10980 tccctatcta ttaatcacca ctcttactgc ccagtcaggtcctcattgtt tcctgaacaa 11040 gagtagatgc tattctttcc actttaagac cttatcctggctggatgcgg tggctcaggc 11100 ttgtaaaccc agcactttgg gaggccgagg caggcagatcacttgaggtc aggagttcaa 11160 gaccagcctg accaacatgg tgaaacccca tctctactaaaaatacaaaa tcagccgggc 11220 gtgtggtgca tgcctgcagt cccagctatt caggtggctgaggcaggaga attgcttgaa 11280 cccaggaggc ggaggttgcg gtgagcctag attgcaccattgcactctag cttgggcaat 11340 agggatgaaa ctccatctca gaagagaaaa gaaaaaaagaccttattctg ttacacaaat 11400 cctctcaatg caatccatat agaataaaca tgtaaccagatctcccaatg tgtaaaatca 11460 tttcaggtag aacagaatta aagtgaaaag ccaagtctttggaattaaca gacaaagttc 11520 aaataacagt cctcatggcc ttaagaattt acctaacattttttttagaa tcaattttct 11580 tatatatgaa ttggaaacat aattcctccc tcacaaacacattctaagat tttaaggaga 11640 tattgatgaa gtacatcatc tgtcattttt aacagttagtggtagtgatt cacacagcac 11700 attatgatct gttcttgtat gttctgttcc attctgtattcttgacctgg ttgtattctt 11760 tctgagctcc agatccacat atctaagtac atctttttgcattttacaag agtgcataca 11820 atacaatgta tccaagactg tatttctgat tttatcgtaccactaaactc acaaatgtgg 11880 ccctattctt gtgttcacga ctgacatcac cgtcatggtccaagtctgat aatagaaatg 11940 gcattgtcac tttcttccct actgcaacag aagcccagctatttgtctcc cattttctct 12000 acttctaaaa tacatttctt cactaagtga gaataatcttttaaagacac aaatcaaacc 12060 atgccaccac ctttcttgaa ttattcaata tctttcgttggcttccaggt tacagaaaaa 12120 taacttgtaa caaagtttaa aggtcattca tggctcctctctaccctatt ttataacatt 12180 tccccttgtg atcagaatct caggcacatc atccatctttctatatacaa ataaagtcat 12240 atagtttgaa ctcacctctg gttactttta atcaaccaaatgctgtaaaa tgcatttgta 12300 tcgctacgtg ttaagcagta gttgattctt ttcatttcttgttaatattc tattctttga 12360 ctataccgta atttatcaat tctactgttg gtaagcatttaagtggctac cggtttgagg 12420 tttttatgat tattgctgtc ataagcattt ctatacatgtctttggatac acacatgcat 12480 gtgtttctga atatctaaaa atgtaattgc taggtaatagacttatcaag catccagcat 12540 ttgtggatac tattaaaggt tttccaaagg ggttatactattgtacagtg tcaccaacag 12600 agtttgagtt tctattgatc catatcacca ccaaaatttgaactgtcagt cttatctctt 12660 ctcttgtctc ttttttcctc ttttttttcc ttcccttcccctctcttcgt ttcttttctc 12720 tcctcttctc ttctttcctc tcttcccttc cctttctctttctcttccct atcccttctc 12780 ctctcctctc ccctcctttt ttctcctctc ctctccattatttatttttc cttcttctcc 12840 tccatccctt ccatcctctc tcttcccctc ttccttccttcctttctcca tttcttcctc 12900 ctctttccct caatccttcc ttttggatat gctcatgggtgtgtatttgt ctgccattgt 12960 ggcattattt gaattcagaa aagagtgaaa aactactgggatcttcattc tgggtctaat 13020 tccacatttt tttttaagaa cacactctgt aaaaatgttctgtactagca tattcccagg 13080 aacttcgtta aatttaatct ggctgaatat ggtaaatctactttgcactt tgcattcttt 13140 ctttagtcat accataattt taaacattca aaatatttgtatataatatt tgattttatc 13200 tgtcattaaa atgttaacct taaaattcat gtttccagaacctatttcaa taactggtaa 13260 ataaacacta ttcatttttt aaatattctt ttaatggatatttatttcaa tataataaaa 13320 aattagagtt ttattatagg aagaatttac caaaagaaggaggaagcaag caagtttaaa 13380 ctgcagcaat agttgtccat tccaacctct caaaattcccttggagacaa aatctctaga 13440 ggcaaagaag aactttatat tgagtcaact tgttaaaacatctgctttta gataagtttt 13500 cttagtataa agtgacagaa acaaataagt taaactctaagatacattcc actatattag 13560 cctaaaacac ttctgcaaaa atgaaactag gaggatatttttagaaacaa ctgctgaaag 13620 agatgcggtg gggagatatg cagaggagaa cagggtttctgagtcaagac acacatgaca 13680 gaacagccaa tctcagggca 13700 3 15 DNA Homosapiens 3 aaacggtccc tggct 15 4 18 DNA Homo sapiens 4 ctccacccatgggttggc 18 5 19 DNA Homo sapiens 5 atttctttct ttctttttt 19 6 20 DNAHomo sapiens 6 tgaatgaatg aacgaatgag 20 7 21 DNA Artificial SequenceDescription of Artificial Sequence gamma-globin forward primer 7tggcaagaag gtgctgactt c 21 8 19 DNA Artificial Sequence Description ofArtificial Sequence gamma-globin reverse primer 8 tcactcagct gggcaaagg19 9 24 DNA Artificial Sequence Description of Artificial Sequencegamma-globin probe 9 tgggagatgc cataaagcac ctgg 24

1. A synthetic double-stranded oligonucleotide having a length comprisedbetween 10 and 50 bases and a nucleic acid sequence selected from thegroup consisting of: a) sequences corresponding to a selected portion ofthe sequence comprised between positions 220 and 290 of SEQ. ID NO: 1;and b) sequences corresponding to a selected portion of the humangenomic region comprised between the γ-globin gene and the δβ-cluster,for use as an inducer of erythroid differentiation.
 2. The syntheticoligonucleotide according to claim 1, characterised in that the nucleicacid sequence of the said human genomic region comprised between the,γ-globin gene and the δβ-cluster is SEQ. ID NO:2.
 3. The syntheticoligonucleotide according to claim 1 or 2, characterised in that it hasa nucleic acid sequence selected from the group consisting of SEQ. IDNO: 3, SEQ ID NO: 4, SEQ. ID NO: 5 and SEQ. ID NO:
 6. 4. The syntheticoligonucleotide according to any of claims 1 to 3, in combination with asecond biological response modifier selected from the group consistingof cytosine arabinoside, retinoic acid, plicamycin, chromomycin,hydroxyurea, guanosine triphosphate (GTP), guanosine diphosphate (GDP),guanosine monophosphate (GMP).
 5. Use of a synthetic double-strandedoligonucleotide according to any of claims 1 to 4, for the manufactureof a medicament for the treatment of β-thalassemia.
 6. Use of asynthetic double-stranded oligonucleotide according to any of claims 1to 4, for the manufacture of a medicament for the treatment ofneoplastic diseases.
 7. A pharmaceutical composition comprising at leasta synthetic double-stranded oligonucleotide according to any of claims 1to 4 and a pharmaceutically acceptable carrier.
 8. A method forpreparing a synthetic double-stranded oligonucleotide according to anyof claims 1 to 3, comprising the steps of; (a) synthesising acorresponding single-stranded oligonucleotide; and (b) annealing thesingle-stranded oligonucleotide with its complementary strand.